Mesenchymal Stem Cells cause Telomere Length Reduction of Molt-4 Cells via Caspase-3, BAD and P53 Apoptotic Pathway

Mesenchymal stem cells (MSCs) as undifferentiated cells are specially considered in cell-based cancer therapy due to unique features such as multi-potency, pluripotency, and self-renewal. A multitude of cytokines secreted from MSCs are known to give such multifunctional attributes, but details of their role are yet to be unknown. In the present study, MSCs were cultured, characterized and co-cultured with Molt-4 cells as acute lymphoblastic leukemia cell line in a trans-well plate. Then, cultured Molt-4 alone and Molt-4 co-cultured with MSCs (10:1) were collected on day 7 and subjected to real time-PCR and Western blotting for gene and protein expression assessment, respectively. Ki-67/caspase-3 as well as telomere length were investigated by flow cytometry and real time-PCR, respectively. The results showed that MSCs caused significant decrease in telomere length as well as hTERT gene expression of Molt-4 cells. Also, gene and protein expression of BAD and P53 were significantly increased. Furthermore, the flow cytometry analysis indicated the decrease and increase of the Ki-67 and caspaspase-3 expression, respectively. It was concluded that MSCs co-cultured with Molt-4 cells could be involved in the promotion of Molt-4 cell apoptosis via caspase-3, BAD, and P53 expression. In addition, the decrease of telomere length is another effect of MSCs on Molt-4 leukemic cells.

esenchymal stem cells (MSCs) are multipotent undifferentiated cells which have a potential to differentiate into different cell types such as adipocytes, osteocytes, neuron like cells etc. (1). Other features of MSCs including self-renewal, plasticity, and non-immunogenic characteristics have led to consider them for cellbased therapy (2). The role of MSCs in some diseases such as cancer, blood disorders, heart failure, genetic and neurodegenerative diseases have been considered in more recent studies (3,4).
Hematologic malignancies have received more attention for cell transplantation with MSCs. The promoting and inhibiting effects of MSCs on cancer cells progression is one of the challenges associated with cell therapy (5,6). Most studies confirmed the inhibitory effects of MSCs on tumor growth, however, only a few have shown stimulatory effects (7). The reason for this contradiction may be related to different types of tumor cells, the heterogeneity of MSCs population, and the number of injected cells (8). Because of these contradictions, further studies are suggested for investigating the precise bidirectional interaction between tumor cells and MSCs. In one study, Zhang and Zhang (2009) indicated that factors such as cytokines which are released from MSCs could inhibit the proliferation rate of chronic myeloid leukemia mononuclear cells (CML-MNCs) in patients (9). In contrast to the study mentioned above, Paino et al. (2018) demonstrated that SAOS2 osteosarcoma and MCF7 breast cancer cell lines are able to maintain adipose tissue-derived MSCs (ADSCs) in a stemnes state, and if these cells persist following surgery, they will most likely induce resident MSCs to boost tumor angiogenesis and proliferation (10). In more detail, the inhibitory/promoting effects of MSCs on The protein expression of signaling pathways components involved in this process, including BCL2 associated agonist of cell death (BAD) and P53, were also evaluated.  (15,16). Primers are presented in Table 1.

Western blotting
Protein expression of BAD and P53 was investigated by Western blotting. For this purpose, cells from control and experimental groups were collected and total cell proteins were extracted.
Next, protein samples were electrophoresed on 12% polyacrylamide gels and transferred to a PVDF membrane. In the following, the membrane was incubated with primary antibodies against BAD and P53, and it was then incubated with corresponding secondary antibodies. The protein bands were detected with X-ray film and GAPDH was used as the internal control to normalize (17,18).

Absolute telomere length (aTL) measurement
aTL was measured using real-time PCR as previously reported by Fathi et al. (2020) (19).
Briefly, DNA was isolated from both control and experimental groups at the end of co-culture period and 20 ng/µl DNA was suitable for aTL measurement. Data obtained from real-time PCR for aTL was analyzed as kb/reaction and genome copies/reaction for telomere and single copy gene.
The primers used for aTL measurment are listed in

Statistical analysis
The results were analyzed using t-test. The statistical significance was determined at P <0.05 by Graph Pad Prism version 6.01.
However, hematopoietic surface markers CD34 (0.67%) and CD56 (0.62%) were not expressed in MSCs. In addition to flow-cytometry, adipogenic and osteogenic differentiation was investigated for multi-lineage differentiation potency of MSCs. As shown in Figure 2, lipid droplets as well as calcium deposits were stained with Oil-red O and Alizarinred, respectively (13).

Investigation of the gene and protein expression of BAD and P53 in Molt-4 cells following coculture with MSCs
For investigating the effect of cytokines secreted from MSCs on Molt-4 cells as acute

Investigation of aTL and hTERT gene expression following co-culture with MSCs
Following the co-culture period, aTL was measured using real-time PCR. As shown in  Data showed that caspase-3 level in the co-cultured group was increased 6.7-fold (Figurs 5F-J).   Without any ethical concerns, MSCs are easily obtained and cell therapeutic strategy using these cells seems to be a better choice for cancers.
However, further researches are needed to use MSCs as clinical application.